The present invention relates to a composition useful as an anti-fouling agent by inhibiting microbial growth, said composition comprising extract and/or its fractions from Zoanthus sp. and optionally de-odorizing agents and a method of producing said extract and its fractions having about 100% anti-bacterial activity fouling microbes.
Chemical antifouling defense strategies of marine organisms have been studied either directly against the target fouling organisms such as bacteria, fungi, algae, invertebrate larvae, etc. (Amade et ai., 1987, Mar. Biol. 94, 271-275; Slattery et al., 1995, J. Exp. Mar. Biol. Ecol, 190, 61-77; Shellenberger and Ross, 1998, Northwest Science, 72(1), 23-33) or using other fouling measures like settlement inhibition, larval moulting rates, photosynthesis, etc. (Kon-ya et al., 1994, Fish. Sci, 60(6), 773-775; Kato et al., 1995, Tetrahedron letters, 36(12), 2133-2136; Mizobuchi et al., 1996, Fisheries Sci, 62(1), 98-100; Becerro et al., 1997, J. Chem. Ecol, 23(6), 1527-1547).
Information on secondary metabolite production marine organisms deals mostly with isolation and characterization of the compounds and very little is known about their ecological and biological importance. In recent years, some attempts have been made in this direction on a few groups such as soft corals, gorgonians, algae, sponges, bryozoans, etc. (Targett, 1988, in Marine Biodeterioration, M. F. Thompson., R. Sarojini., R. Nagabhushanam., A A Balkema eds, Rotterdnm, 607-617; Standing et al., 1984, J. Chem. Ecol, 10, 823-824; Davis et al., 1991, Mar. Ecol, Prog. Ser, 72, 117-123; Vitalina et al., 1991, in Bioactive compounds from marine organisms, M. F. Thompson., R. Sarojini., R. Nagabhushanam eds, Oxford and IBH Publishing Co. Pvt. Ltd, 63-68; Szewzyk et al., 1991, Mar. Ecol. Prog. Ser, 75, 259-265) leaving many other groups unstudied. Zoanthid (Phylum Coelenterata; Family Zoanthidae) is one such group which has received very little attention with respect to chemical defense strategies.
Zoanthids are benthic, sessile, colony forming organisms, comprising of thousands of individual polyps. Several chemical compounds have been isolated from zoanthids, possessing various pharmacological properties,(Moore and Scheuer, 1971, Science, 172, 495-498; Cimino et al., 1973,; Sturaro et al., 1982; Rao et al., 1985, J. Org. Chem, 50, 3757-3760; Bakus et al., 1986, J. Chem. Ecol, 12, 951-987; Quinn, 1988, in Bioorganic Marine Chemistry, P. J. Scheuer ed. Vol 2, 25-27; Shigemori et al., 1999, J. Nat. Prods, 62(2), 373). However, very little is known about the functions of these metabolites in their natural habitat. In the Anjuna beach of Goa (west coast of India), they occur as dense mats and colonise a large intertidal area. The main focus of the present Invention{circumflex over ( )} therefore, is to ujidjijtjadj{circumflex over ( )}e{circumflex over ( )}h{circumflex over ( )}nucal antifouling defense properties against common micro-fouling organisms viz. bacteria and diatoms utilising the extract of Zoanthus sp and in turn for identifying a natraJ product useful in antifouling technqlojgy.
Any surface exposed to marine environment undergoes sequential changes that begins with the adsorption of a layer of bio-polymers followed by the formation of a bacterial film, growth of pinnate diatoms and protozoans (Baier, 1984, in Marine Biodeterioration: An Interdisciplinary Study, J. D. Costlow and R. C. Tipper, ed, Naval Institute Press, Anapolis, Md., 57-62; Mitchell and Kirchman, 1984, in Marine Biodeterioration: An Interdisciplinary Study, J. D. Costlow and R. C. Tipper, ed, Naval Institute Press, Anapolis, Md., 49-56; Wahl, 1989, Mar. Ecol. Prog. Ser, 58, 175-189). Consequently, the variety of bacterial species forming the microbial film may influence, inhibit or at times have no effect upon the later settling species which constitute the invertebrate larvae that settle and metamorphose into adults (Bakus et al., 1986, J. Chem. Ecol, 12, 951-987). In most cases where it has been shown that a microbial film is necessary for settling of larvae and algal propagules, inhibition of bacterial growth would prevent the microbial film formation and in turn inhibit larval settlement, ultimately leaving the surfaces free of epibionts.
In this effort, the test animals were collected and the crude methanol extract was prepared. The antifouling potential of the extract was then explored against known fouling diatoms and bacteria in the laboratory. After testing the activity of the crude extract, it was fractionated using solvents of increasing polarity. These fractions were tested against the fouling diatoms. The results of the evaluation reveals that the extract of Zoanthus sp has the antifouling property.
The main object of the present invention is to develop a composition comprising an extract from Zoanthus species.
Another main object of the present invention is to develop a composition having anti-fouling property.
Yet another object of the present invention is to develop fractions from extract of animal Zoanthus species.
Still another object of the present invention is to fractionate the crude extract using solvents of increasing polarity, viz; petroleum ether, chloroform, n-butanol and the aqueous fraction.
Still another object of the present invention is to identify the fraction which is most effective hi against fouling diatoms.
Still another object of the present invention is to develop a method of preparing composition comprising an extract from Zoanthus species.
Still another object of the present invention is to develop a method of preparing said composition having 100% anti-bacterial activity.
Still another object of the present invention is to develop a method of preparing said composition having 100% microbial growth inhibitory activity against fouling microbes.
Still another object of the present invention is to develop a method of preparing said composition with anti-microbial activity wherein, said activity is particularly effective in marine environment.
Still another object of the present invention is to develop a method of preparing said composition with anti-microbial activity wherein, said activity is particularly effective against bacteria and diatoms.
The present invention relates to a composition useful as an anti-fouling agent by inhibiting microbial growth, said composition comprising extract and/or its fractions from Zoanthus sp. and optionally de-odorizing agents and a method of producing said extract and its fractions having about 100% anti-bacterial activity fouling microbes.
Accordingly, the present invention relates to a composition useful as an anti-fouling agent by inhibiting microbial growth, said composition comprising extract and/or its fractions from Zoanthus sp. and optionally de-odorizing agents and a method of producing said extract and its fractions having about 100% anti-bacterial activity fouling microbes.
In one embodiment of the present invention, a composition comprising extract and/or its fractions from Zoanthus sp, and optionally de-odorizing agents wherein said composition is useful as an anti-fouling agent against microbes.
In another embodiment of the present invention, wherein said extract and its fractions are prepared by using either polar or non-polar solvent.
In yet another embodiment of the present invention, wherein animal species preferably used for said activity is Coelenterata Zoanthidea. 
In still another embodiment of the present invention, wherein said composition is prepared by using whole body tissue of said species.
In still another embodiment of the present invention, wherein said de-odorizing agent shows no affect on the activity of said extract and fractions.
In still another embodiment of the present invention, wherein ratio of said extract and/or fractions and de-odorizing agent is ranging between 1:1 to 1:10.
In still another embodiment of the present invention, wherein said composition is particularly effective against bacteria and diatoms.
In further embodiment of the present invention, a method of producing an extract and its fractions from Zoanthus sp, useful as an anti-fouling agent by inhibiting microbial growth.
In another embodiment of the present invention, soaking washed specimen of said species in methanol.
In yet another embodiment of the present invention, obtaining crude extract from the whole body tissue of said species.
In still another embodiment of the present invention, decanting the solvent extract.
In still another embodiment of the present invention, filtering decanted extract.
In still another embodiment of the present invention, evaporating the filtrate at 40xc2x0 C. to obtain concentrate crude extract.
In still another embodiment of the present invention, fractionating concentrated crude extract using solvents of increasing polarity.
In still another embodiment of the present invention, introducing said crude extract at concentration ranging between 0.1 to 5.0-mg/6 mm-diameter disc in methanol on sterilized filter paper disc.
In still another embodiment of the present invention, evaporating the solvent from the disc.
In still another embodiment of the present invention, exposing microbes to said extract.
In still another embodiment of the present invention, laying said disc on agar plates seeded with test bacterium.
In still another embodiment of the present invention, running a control with solvent not containing extract.
In still another embodiment of the present invention, incubating said plates overnight for temperature ranging between 25 to 32xc2x0 C.
In still another embodiment of the present invention, measuring growth inhibition zone.
In still another embodiment of the present invention, determining % inhibition of microbes using said extract.
In still another embodiment of the present invention, determining anti-diatomic activity of crude extract and said fractions separately, by placing diatoms with initial cell suspension of concentration ranging between 100 to 5000 cells/ml in flasks.
In still another embodiment of the present invention, adding said crude extract of concentration ranging between 10 to 5000 ppm to said cell suspension.
In still another embodiment of the present invention, adding said fractions of concentration ranging between 10 to 500 ppm in separate flasks.
In still another embodiment of the present invention, incubating flask at temperature ranging between 25 to 32xc2x0 C. with 12 hrs light and next 12 hrs dark exposure conditions.
In still another embodiment of the present invention, determining % growth inhibition after about 6 days i.e., during exponential growth phase of diatoms.
In still another embodiment of the present invention, wherein said species preferably used for said activity is Coelenterata Zoanthidea. 
In still another embodiment of the present invention, wherein said method is particularly effective in marine environment.
In still another embodiment of the present invention, wherein said method is particularly effective against bacteria and diatoms.
In still another embodiment of the present invention, wherein soaking said washed specimen in methanol for number of days ranging between 5-15.
In still another embodiment of the present invention, wherein said species is obtained from intertidal rocks.
In still another embodiment of the present invention, wherein decanted extract is filtered through whatman filter paper I.
In still another embodiment of the present invention, wherein filtrate is evaporated using vacuum.
In still another embodiment of the present invention, wherein said method uses zobell marine agar for bacterial culture.
In still another embodiment of the present invention, wherein concentration of said extract against bacteria is preferably about 0.5 mg/mm of diameter disc.
In still another embodiment of the present invention, wherein said extract is incubated at preferably 28xc2x0 C.
In still another embodiment of the present invention, wherein growth inhibition zone less than 1 mm not been scored.
In still another embodiment of the present invention, wherein said fractionating solvents are selected from a group comprising petroleum ether, chloroform, n-butanol, and aqueous.
In still another embodiment of the present invention, wherein dissolving non-polar fraction petroleum ether in acetone (0.005-0.01 ml).
In still another embodiment of the present invention, wherein bacteria are selected from a group comprising Bacillus cereus, B. circulans, Pseudomonas vesicularis, and P. putida. 
In still another embodiment of the present invention, wherein antibacterial activity of extract is 100%.
In still another embodiment of the present invention, wherein diatoms are selected from a group comprising Navicula subinflata and Nitzschia closterium. 
In still another embodiment of the present invention, wherein calculating % growth inhibition with extract and/or fractions having more than 50% growth inhibition is considered as active ones.
In still another embodiment of the present invention, wherein said extract shows 100% inhibition in diatoms at concentration of 350 ppm and above.
In still another embodiment of the present invention, wherein all the said fractions of crude extract show inhibitory activity against diatoms.
In still another embodiment of the present invention, wherein petroleum ether fraction is most effective of all the fractions against diatoms.
In still another embodiment of the present invention, wherein petroleum ether fraction shows 100% inhibition at concentration of about 80 ppm and above against diatoms.
In still another embodiment of the present invention, wherein chloroform fraction shows about 100% inhibition in diatoms at concentration about 200 ppm.
In still another embodiment of the present invention, wherein n-butanol fraction shows about 100% inhibition against diatoms at concentration ranging between 150-250 ppm.
In still another embodiment of the present invention, wherein aqueous fraction shows inhibitory activity ranging between 70-85% against diatoms at concentration ranging between 140-250 ppm.
In still another embodiment of the present invention, wherein said extract/fractions do not have any toxic/adverse effect on non-target organisms.
In still another embodiment of the present invention, wherein assaying microbes in a culture media using agar plate diffusion method.
In still another embodiment of the present invention, wherein said method is used to prevent fouling in food articles.
In still another embodiment of the present invention, wherein said method is used to prevent fouling in biological specimens.
In further embodiment of the present invention, the present invention provides a process for the preparation of antifouling crude extract from Zoanthus sp and the said process comprising,
i) Collection of live/fresh specimens of Zoanthus sp from intertidal rocks.
ii) Washing the specimens clean with fresh sea water and soaking in methanol for
about 10 days iii) Filtering the extract through Whatman filter paper I and vacuum evaporating
at 40UC to concentrate to crude residue. In an embodiment, the crude extract of Zoanthus sp is exhibiting antibacterial property.
In a preferred embodiment, the crude extract of Zoanthus sp is inhibiting growth of fouling bacteria such as Bacillus cereus, Bacillus circulans, Bacillus pumilus, Pseudomonas vesiciulans and Pseudomonas putida. 
In another embodiment, the crude extract of Zoanthus sp is inhibiting growth of fouling diatoms at all concentrations.
In a preferred embodiment, the crude extract of Zoanthus sp is completely inhibiting growth of diatoms, Navicula subinfiata at and above 600 ppm concentration and Nitzschia closterium at and above 400 ppm concentration.
In still another embodiment of the present invention, a process for fractionating the crude antifouling extract from Zoanthus sp based on increasing polarity of the solvents, viz. petroleum ether, chloroform, n-butanol and aqueous fractions and testing the antifouling property of all these fractions against the fouling diatoms Navicula subinflata and Nitzschia closterium, the said process at comprising, obtaining crude extract from livefreshly collected Zoanthus sp, washing them clean with sea water, soaking in methanol for about 10 days, filtering the extract through Whatman filter paper I and concentrating to crude residue under vacuum at 40xc2x0 C.
In an embodiment, the crude extract of Zoanthus sp is fractionated based on increasing polarity of solvents into petroleum ether, chloroform, n-butanol and aqueous fractions.
In another embodiment, all four fractions of the crude extract of Zoanthus sp are inhibiting the growth of fouling diatoms, Navicula subinflata and Nitzschia closterium. 
In yet another embodiment, the percentage inhibition in all the fraction is varying with concentrations.
In still another embodiment, the petroleum ether fraction of the crude extract of Zoanthus sp is more effective in inhibiting growth of the fouling diatoms Navicula subinflata and Nitzschia closteriwn. 
In one more embodiment, the petroleum ether fraction of the crude extract of Zoanthus sp completely inhibits growth of Navicula subinflata at and above 160 ppm and Nitzschia closterium at and above 80 ppm concentrations.
In further embodiment of the present invention, a process for the preparation of an antifouling crude extract from Zoanthus sp and testing the said crude extract on fouling bacteria and diatoms.
The invention also relates to a process for fractionation of the crude extract and testing the antifouling property of the fractions on fouling bacteria.
A process for the preparation of antifouling crude extract from Zoanthus sp and the said process comprising,
Collection of live/fresh specimens of Zoanthus sp from intertidal rocks.
Washing the specimens clean with fresh seawater and soaking in methanol for about 10 days.
Filtering the extract through Whatman filter paper I and vacuum evaporating at 40xc2x0 C. to concentrate to crude residue.
In still another embodiment of the present invention, wherein the crude extract of Zoanthus sp is exhibiting antibacterial property.
In still another embodiment of the present invention, wherein the crude extract of Zoanthus sp is inhibiting growth of fouling bacteria such as Bacillus cereus, Bacillus circulans, Bacillus pumilus, Pseudomonas vesiciularis and Pseudomonas putida. 
In still another embodiment of the present invention, wherein the crude extract of Zoanthus sp is inhibiting growth of fouling diatoms at all concentrations.
In still another embodiment of the present invention, wherein the crude extract of Zoanthus sp is completely inhibiting growth of diatoms, Navicula subinflata at and above 600 ppm concentration and Nitzschia clostenium at and above 400 ppm concentration.
In still another embodiment of the present invention, wherein-the crude extract of Zoanthus sp may have potential use as an antifouling agent/in aquatic environment.
In still another embodiment of the present invention, a process for fractionating the crude antifouling extract from Zoanthus sp based on increasing polarity of the solvents, viz. petroleum ether, chloroform, n-butanol and aqueous fractions and testing the antifouling property of all these fractions against fouling diatoms, the said process comprising, obtaining crude extract from live/freshly collected Zoanthus sp, washing them clean with sea water, soaking in methanol for about 10 days, filtering the extract through Whatman filter paper I and concentrating to crude residue under vacuum at 40xc2x0 C.
In still another embodiment of the present invention, wherein the crude extract of Zoanthus sp is fractionated based on increasing polarity of solvents into petroleum ether, chloroform, n-butanol and aqueous fractions.
In still another embodiment of the present invention, wherein all the four fractions of the crude extract of Zoanthus sp are inhibiting the growth of fouling diatoms, Navicula subinflata and Nitzschia closterium. 
In still another embodiment of the present invention, wherein the percentage inhibitions in all the fraction is varying with cone.
In still another embodiment of the present invention, wherein the petroleum ether fraction of the crude extract of Zoanthus sp is more effective in inhibiting growth of the fouling diatoms Navicula subinflata and Nitzschia closterium. 
In still another embodiment of the present invention, wherein the petroleum ether fraction of the crude extract of Zoanthus sp completely inhibits growth of Navicula subinflata at and above 160 ppm and Nitzschia closterium at and above 80 ppm concentrations.
In still another embodiment of the present invention, wherein all fractions of the crude extract of Zoanthus Sp, more particularly the petroleum ether fraction may have potential use as an antifouling agent in the aquatic environment.
The invention is further explained with the help of the following examples and should not be construed to limit the scope of the invention.